Evaluation of hematopoietic stem cell expansion in the presence of garcinol.

Objective
The application of human cord blood (hCB) is limited to children by using relatively small volume of cord blood that does not contain enough hematopoietic stem cells (HSCs). So, efforts for applying cord blood stem cells in transplantation have led to establishment of some approaches for ex vivo expansion of HSCs such as garcinol.


Materials and Methods
CD133+ HSCs were separated by a magnetic-activated cell sorting (MACS) system. Isolated cells were cultured with different doses of garcinol, SCF, TPO and FLT-3L. The optimal dose of garcinol for ex vivo expansion of HSCs was determined by direct counting. Flow cytometry was used to evaluate the expression of CD133 marker to check the ability of garcinol in maintenance of HSCs. Colony forming cell (CFC) assay was performed to evaluate clonogenic capability of treated cells. The level of expression of CXCR4 gene was evaluated by RT-PCR. Data were analyzed using Student's t test.


Results
Our results showed that CD133+ HSCs in the presence of garcinol (5-10 µM) had high expansion activity and cell counting showed that the number of cells in treated group was higher than control group (1.9 -fold) and CFC assay showed that the number of colonies following treatment with garcinol had 1.3-fold increase. Treatment of HSCs with garcinol resulted in 9.6-fold increase in terms of CXCR4 expression in comparison to control group.


Conclusion
The present study showed that garcinol can improve ex vivo expansion of HSCs and enhance their potential for homing to bone marrow.


Introduction
In recent years, autologous or allogeneic transplantation of hematopoietic stem cells (HSCs) has been performed as a medical approach for a number of diseases such as various blood diseases attributed to hematopoietic and immune dysfunctions as well as leukemias (Kondo et al., 2003;Verma et al., 2005).
Human cord blood is an unlimited and unique source of HSCs (Broxmeyer et al.,1989). Umbilical cord blood (UCB) transplants have been done worldwide in pediatric and adult patients (Gluckman et al., 2004). UCB stem cells are an interesting alternative to adult BM and mobilized peripheral blood (PB) stem cell. It has been shown that significant functional differences exist between UCB-derived HSCs and adult BM-derived HSCs. UCBderived HSCs have a higher proliferation and expansion potential, which might be associated with their greater telomere length (Holyoake et al., 1999;Piacibello et al., 1997) and their ability to exit G0-G1 more rapidly (Mayani et al.,1998;Traycoff et al.,1994).
UCB-derived HSCs have been collected by a simple procedure with no danger for the mother or the newborn; in addition, these cells are less contaminated with blood-borne viruses than adult stem cell sources. The most important advantage of UCB is that there is no need to full human leukocyte antigen (HLA) compatibility and there is a lower risk of graft versus host disease (GvHD) in comparison to other sources because of low number of lymphocytes in UCB (Broxmeyer et al.,1989;Jaroscak et al.,2003;Wang et al.,1998).
However, a single UCB unit contains a lower number of HSCs as compared to other sources (Gluckman et al.,2004;Cohen et al.,2004). This is an important limiting factor when adult recipients undergo transplantation.
To overcome this difficulty, various approaches have been used including transplantation of two UCB units (double umbilical cord blood transplantation, dUCBT) (Sideri et al.,2011), and intra-marrow injection (Frassoni et al.,2010) to improve the homing efficiency of the transplanted HSCs, or ex vivo expansion of HSCs (Jaroscak et al.,2003;De Lima et al.,2008;Kelly et al.,2009). However, double cord transplantation might be associated with increased risk of GvHD and might not significantly reduce the time of neutrophil and platelet engraftment. In addition, intra-bone injection -although it was shown to be safedid not shorten the time of neutrophil engraftment (Brunstein et al.,2009). For ex vivo expansion of HSCs, cell culture systems have used growth factors like stem cell factor (SCF), thrombopoietin (TPO), fms-like tyrosine kinase-3 ligand (FLT-3L), interleukin 6 (IL-6), Notch ligand Delta1, angiopoietin-like proteins, and pleiotrophin. However, protein-factor combinations have proven to be neither affordable nor readily available. Smallmolecule compounds (SMCs) have been played vital roles in molecular biology and pharmaceutical therapy. The use of SMCs has also improved our understanding of signaling pathways that control stemness. Increasing the knowledge in this field can help researchers to promote current methods used for ex vivo expansion of HSCs (De Lima et al.,2008;Ding et al.,2004;Boitano et al.,2010).
Garcinol, a benzophenone derivative originally isolated from Garcinia indica, is one of the SMCs that can activate signaling pathways that promote HSC expansion (Krishnamurthy et al.,1981;Mantelingu et al.,2007). In this study, we evaluated the effect of garcinol on expansion of UCBderived CD133 + HSCs.

Preparation of the extracts
Garcinol was obtained from Santa Cruz Biotechnology. Garcinol was stirred for 10 hr in DMSO at room temperature, and placed for 17 hr at 4°C. Then, the extract was filtered and stored at -20℃.

Determination of the optimal dose of Garcinol
To identify the optimal dose of garcinol that exerts the optimum effect on expansion of HSCs. CD133 + HSCs were cultured with different doses of garcinol in the presence of SCF, TPO and FLT-3L (Stem Cell Technology). After 8 days, cells were stained with trypan blue and viable cells were counted.

Flow cytometry
The percentage of CD133 + HSCs was assayed by flow cytometry (ATTUNE ® Flow cytometer (Applied Biosystems), Stem Cell Technology Research Center, Tehran, Iran) on days 0, 6 and 11. Treated and non-treated HSCs were incubated with phycoerythrin (PE)-conjugated anti-CD133 antibodies (Miltenyi Biotec) according to the manufacturer's instructions.

Colony forming cell (CFC) assay
The potential of HSCs to proliferate and differentiate into colonies was evaluated before and after expansion with garcinol by colony forming cell (CFC) assay. About 1000-1500 cells were mixed in 2 ml of methocult H4435 (Stem Cell Technology) containing 50 ng/mL human SCF, 20 ng/mL FLT-3L, and 20 ng/ml TPO. The number of colonies which were incubated at 37 °C for 14 days, was counted by using an inverted microscope.

Real-time reverse transcription PCR and Gene expression analysis
UCB-derived CD133 + HSCs were isolated by cell sorting with MACS. CD133 + HSCs were cultured in the presence of garcinol or DMSO for 11 days. Total RNA of the isolated cells was extracted on days 0, 6, and 11. Total RNA extraction, cDNA synthesis, real-time PCR and calculation of gene expression were performed as previously described (Anbarlou et al.,2015). The PCR primer sequences used for real-time PCR in this study, are shown in Table 1.

Statistical analysis
All tests and analyses were conducted in triplicate. Data means were compared using Student's t test. Statistical analyses were performed by SPSS version 16.0 and GraphPad Software (GraphPad PRISM V 5.0).

Results
Determination of the optimal dose of garcinol CD133 + HSCs were treated with different doses of garcinol in the presence of SCF, TPO, and FLT-3L for 8 days to find the optimal dose of garcinol that has the best effect on HSCs expansion. The results showed that CD133 + HSCs in the presence of garcinol 5-10 µM had high expansion activity (Figure 1). In accordance with the study of Taito Nishino et al. (2011), the 10µM concentration of garcinol was selected for the experiments.

Garcinol improves the expansion of human hematopoietic Stem Cell
To recognize biologically-active natural products that affect ex vivo expansion of HSCs, hCB-derived CD133 + HSCs were cultured with garcinol (10 µM) in the presence of SCF, TPO, and FLT-3L. After 11 days, the number of CD133 + HSCs was enumerated (Figure 2). Cell counting showed that the number of cells in treated group was higher than control group (1.9fold) (control cells were treated with DMSO to remove the possible effect of drug solvent). Figure 1. Determination of the optimal dose of garcinol. The results demonstrated that the best expansion activity of HSCs was achieved in the presence of 5 or 10 µM of garcinol. (Mean±SEM, n=3). *p<0.05, **p<0.01, and *** p<0.001.

Flow cytometry results
The purity of CD133 + HSCs was evaluated by flow cytometry on days 0, 6 and 11. The percentage of CD133 + cells was nearly 92% after immunomagnetic separation (Figure 3). The percentage of CD133 marker in HSCs in control group was 50% and 23% on days 6 and 11, respectively and in treated group, it was 76% and 36% on days 6 and 11, respectively (Figure 4).

Hematopoietic colony forming cell assay
Colony forming cell (CFC) assay showed that the number of colonies in cells treated with garcinol increased (on average 1.3-fold; p=0.01) ( Figure 5). These data demonstrated that garcinol can promote colonogenic capacity of HSCs. When cells were separated from body, they had started asymmetrical division in vitro. Thus, some of them had been differentiated into progenitor cells which decreased their colonogenic capacity. So, the number, percentage and the colonies of the untreated cells reduced by increasing time. Garcinol could partially prevent this outcome.

Effects of garcinol on CXCR4 gene expression
CXCR4, a chemokine receptor, has an important role in quiescence and homing of HSCs. Treatment of HSCs with garcinol for 11 days led to up-regulation of CXCR4 in cells. Expression levels of CXCR4 were measured by qRT-PCR. Treatment of HSCs with garcinol resulted in 9.6-fold increase in CXCR4 expression in comparison to control group (Figure 6). show statistically significant differences. Figure 6. Effects of garcinol on CXCR4 expression. Bar graph shows relative expression levels of CXCR4 gene. The relative expression levels of CXCR4 in HSCs treated with garcinol was increased (9.6-fold; p<0.001) in comparison to control group; Values are shown as mean±SEM. *p<0.05, **p<0.01, and ***p<0.001 show statistically significant differences.

Discussion
Our results showed that garcinol (10 µM) markedly increased the expansion of HSCs as compared to control. In addition, colony formation assay showed 1.3-fold increase in colony formation in HSCs treated with garcinol. Treatment of HSCs with garcinol resulted in 9.6-fold increase in CXCR4 expression in comparison to control group. Furthermore, garcinol may enhance HSCs homing by up-regulating the expression of CXCR4 gene. Garcinol or camboginol, is a poly isoprenylated benzophenone derivative which is isolated from Garcinia indica (Krishnamurthy et al.,1981).
Garcinol was applied for ex vivo expansion of HSCs because of its ability in inhibition of histone acetyltransferases (HAT 300). Also, garcinol reduces the level of p53 acetylation. p53 limits the selfrenewal of HSCs (TeKippe et al., 2003). Increased expression of Bmi-1 promotes HSCs self-renewal. Overexpression of Bmi-1 enhances symmetrical division of HSCs by increasing the transcriptional repression of tumor suppressor genes, such as Ink4a and Arf (Iwama et al., 2004).
Garcinol has several biological activities, specifically anticancer potential which is mediated via its anti-oxidative, anti-inflammatory, anti-angiogenic, and pro-apoptotic activities. The beneficial properties of garcinol are dose-dependent (Saadat et al.,2012). There are few articles that have focused on the effect of garcinol on ex vivo expansion of HSCs. First, Taito Nishino et al. (2011) introduced garcinol as a novel stimulator of hCB-derived HSCs expansion (Nishino et al., 2011). HSCs can be isolated based on CD34 or CD133 cell surface markers. Early primitive HSCs do not express CD34 marker, so we applied CD133 marker for isolation of HSCs (Mossahebi et al., 2017). Taito Nishino et al. (2011) isolated HSCs by using anti-CD34 antibody. They reported that garcinol (10 µM) increased the numbers of CD34 + CD38 -HSCs by more than 4.5-folds (Nishino et al., 2011), while in the current study the number of CD133 + cells were increased by more than 1.9-folds. They only evaluated the expansion of HSCs and did not address the engraftment potential of expanded cells.
It is thought that garcinol acts in a similar way to that of Bmi1 by inhibiting the activity of histone acetyltransferases (Konuma et al.,2010). In addition to garcinol, other SMCs have been found to be effective on HSCs expansion by inhibiting components of the epigenetic machinery (Nishino et al.,2012;Ko et al.,2011). It seems that an important limitation of garcinol usage is the close proximity of toxic doses and non-toxic efficient doses. More studies in animals will certainly reveal the quality of expanded cells.
The present study showed that garcinol can improve ex vivo expansion of HSCs and enhance their potential for homing to bone marrow. We suggest that a combination of SMCs with different methods may improve the efficacy of current methods for ex vivo expansion of HSCs.